Hsp90 is a unique chaperone that is essential in eukaryotes and that helps to produce and maintain the active state of a select set of biologically and medically important substrates/clients including many signal transduction proteins. Through these clients, Hsp90 is involved in biological processes including aging, signal transduction and evolution. Hsp90 function requires ATP hydrolysis and the dynamic binding and release of clients and numerous co-chaperones. This type of dynamic macromolecular assembly process underlies many critical biological processes including DNA replication and the initiation of transcription. Understanding the conformational dynamics of Hsp90 will provide insights into other dynamic macromolecular complexes and determine the role of chaperones in signal transduction. Many different conformational cycles of Hsp90 are possible based on the biochemical properties of Hsp90. We are elucidating the biologically relevant Hsp90 conformations in vivo. We use protein engineering strategies to thermodynamically stabilize Hsp90 in distinct conformations in order to determine their biochemical properties and their function in vivo. The results of these experiments will delineate the Hsp90 conformations that activate clients in vivo and determine the biologically relevant Hsp90 chaperone cycle. In conjunction with our in vivo studies, we are developing FRET experiments to monitor the kinetics of Hsp90 conformational changes during client maturation. Hsp90 is a structurally flexible homodimer that contains two dimerization domains: the C-domain is predominantly dimeric at physiologic concentration, while the N-domain is the site of ATP hydrolysis and forms transient dimers, There are two aims to this proposal: (1) to determine the role of N-domain association in the Hsp90 chaperone cycle and the activation of substrates, and (2) to elucidate the function of each Hsp90 subunit during the activating substrates. The powerful combination of in vivo experiments and protein engineering together with thermodynamic and kinetic analyses will provide unique insight into the mechanism of Hsp90.